Atherosclerotic cardiovascular disease is a leading cause of morbidity and mortality in the industrialized western hemisphere. Coronary artery disease, the pathologic process of arterial luminal narrowing by atherosclerotic plaque resulting in obstruction of blood flow to the heart, accounts for about half of the deaths. Although catheter-based revascularization or surgery-based treatment approaches have been successful in restoring blood flow to ischemic myocardium in the majority of cases, the treatments are inadequate for a significant number of patients who remain incompletely revascularized. The ramifications of treatment limitations may be significant in patients who have large areas of ischemic, but viable myocardium jeopardized by the impaired perfusion supplied by vessels that are poor targets for conventional revascularization techniques. Treatment alternatives, including mechanical approaches such as percutaneous transluminal myocardial revascularization, and the like, have not produced encouraging results. Gene therapy using adenoviral vectors to augment cytokine production and, therefore, promote angiogenesis has shown promise, but this therapy has limitations and has not yet emerged as the optimal treatment for these patients. Therefore, therapeutic angiogenesis has attracted many researchers attempting to discover a way to circumvent the burden of chronic myocardial ischemia.
Atherosclerosis of the extremities is a leading cause of occlusive arterial disease of the extremities in patients over age 40. Peripheral vascular occlusive disease and its complications, including ulcers and even necrosis of the affected limb, is also common. Although percutaneous transluminal angioplasty and aorto-bifemoral bypass procedures are associated with acceptable morbidity and mortality risk and are usually initially successful, these interventions have not been shown to be effective long-term.
In an effort to provide treatment for myocardial ischemia and/or peripheral vascular occlusive disease, a number of angiogenesis techniques are now in clinical trial, including gene therapy and the use of growth factors such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) to induce or augment collateral blood vessel production. For optimal therapeutic outcome, these techniques rely on the availability of a resident population of mobilizable and hormone responsive vascular endothelial cells in the patient's circulation. However, an age-related diminution of vascular endothelial cell number and function has been observed in adults. In particular, in older patients who are most likely to suffer from vascular problems, both central (i.e. coronary) and peripheral, the number of hormone responsive endothelial cells is reduced and the number of dysfunctional endothelial cells is increased. Moreover, administration of cytokines to mobilize sufficient patient-derived responsive cells may worsen cardiovascular pathophysiology secondary to leukocytosis and/or activation of pro-coagulant processes.
Therefore, an alternative therapy, that of supplying an exogenous source of endothelial precursor cells (EPCs), may be optimal for cellular therapeutics to enhance vasculogenesis and collateralization around blocked/narrowed vessels to relieve ischemia. Clinical use of autologous patient-derived sources of stem cells is advantageous to avoid potential adverse allogeneic immune reactivity; however, the disadvantages include the need to subject the patient to stem cell collection at a time of active vascular disease.
Therefore, there is still a need to develop treatment modalities for both myocardial ischemia and peripheral vascular disease that can promote vasculogenesis in the ischemic tissue.
Transfusion of incompatible ABO blood units is the major cause of transfusion-induced fatalities.14,15 An automated red blood cell production system where the majority of units are group O (Universal donor) could theoretically solve many complications.16
Advanced medical support requirements for military personnel have become an ever growing challenge with the advent of smaller more widespread deployments in the war on terrorism. A recurring theme in management of the advanced theater trauma patient is the pressing need to replenish the patient's circulating volume of blood with universal donor oxygen carrying erythrocytes in order to improve survival. Applicants disclosures relating to erythrocyte progenitor manufacturing technology that will result in a technologically feasible manufacturing solution to the pressing need for constant supply of universal donor red blood units in advance military operations. There is an increasing interest in alternative sources of transfusable blood products due to tighter regulations imposed that leads to increasingly stringent eligibility criteria for blood donors, Thus, there is an ongoing interest in the field to produce fully functioning red blood cells ex vivo. New blood substitutes based on cell-free hemoglobin solutions have had serious problems during clinical trials 21. Moreover, cellular hemoglobin-based blood substitutes are not suitable due to their short circulatory life span of less than 48 hours, compared to 42-120 days for human red blood cells.
It has been well documented that hematopoietic stem cells generate progenitor cells that undergo terminal differentiation, resulting in mature circulating blood cells. 22. Prior culturing techniques for expanding stem cells use a variety of approaches including co-culture with bone marrow stromal cells which rarely show more than 3-5 fold increase in cell number. Clinical grade media-only based culturing systems are inefficient due to the requirement for multiple media changes, and have demonstrated difficulty maintaining the starting populations in undifferentiated states. In addition, co-culture methods have not been shown to effectively produce clinical grade stem cells.23-27 However, the failure of erythroid development, which normally culminates in mature enucleated reticulocytes and red blood cells in vitro or ex vivo is articulated to the need for cell-cell interactions. 6-11 Recently, Giarratana et al 12 describe a promising technology that permits for the first time both the extensive expansion of CD34+ stem cells and their total conversion ex vivo into mature enucleated red blood cells. Although the work of Giarratana et al. is a significant milestone in red blood cell engineering, its practical implications are limited due to the constraints in obtaining enough erythrocyte progenitor cells using the co-culture techniques. In addition, the complex engineering needed to implement the three-step protocol would make each unit of blood produced prohibitively expensive. Thus, developing a practical method for producing red blood cells on a scale that is robust and economical would be ideal.